The experiments in this project are aimed at defining specific pathways for the replication and integration of bacteriophage Mu DNA. Phage Mu is unique among prokaryotic viruses because the physiology of its developmental cycle closely resembles what is known about the replication and integration of animal tumor virus DNA. Experiments designed to evaluate the hypothesis that Mu integrates its genome at the chromosomal replication forks of its host are described. These include the development of a synchronized cell system that provides an immediate assay for the integration of newly replicated phage progeny molecules and the use of density-labeling techniques to elucidate the nature of the integration target site. The question of whether the integrative form of Mu DNA is single-stranded or double-stranded will be studied by a combination of techniques, including electron microscopy, restriction endonuclease analysis, and DNA-DNA hybridization methods. Since the integrative form of Mu is very likely to be an immediate product of viral DNA replication, this project also seeks to identify the replicative form(s) of Mu. Phage Mu DNA replication will be studied by analyzing infecting viral DNA for changes in its conformation and structure in both normal host cells and in cells in which chromosome replication is arrested. The latter system may facilitate the physical isolation of replicating viral DNA molecules by forcing Mu to replicate extrachromosomally.